DESCRIPTION: This research project is directed toward elucidating the structure, function, and regulation of the mammalian pyruvate dehydrogenase (PDH) multi-enzyme complex. The PDH complex is localized to the mitochondria, where it plays a critical role in regulating cellular fuel utilization. The activity of the mammalian PDH complex is regulated primarily by phosphorylation and dephosphorylation, catalyzed by a specific PDH kinase and a PDH phosphatase, respectively. PDH phosphatase (PDP) is a heterodimer consisting of a Mg2+-dependent and Ca2+-stimulated catalytic subunit (PDPc) and a flavoprotein (PDPr). PDH phosphatase activity is stimulated by insulin in several tissues, particularly in adipose tissue. A critical outstanding issue is how the signal is transferred from the insulin receptor in the plasma membrane to the PDH phosphatase within the mitochondrial membrane-matrix compartment. Recent observations suggest that PDPr may be involved in this signaling pathway. The proposed studies seek to elucidate the nature and regulation of the mammalian PDH phosphatase and the macromolecular organization of the Saccharomyces cerevisiae PDH complex, which is a good model for the mammalian PDH complex. The specific aims are: (1) elucidate the nature and function of the regulatory subunit (PDPr) of bovine PDH phosphatase and (2) determine the number and localization of binding sites for the E3-binding protein and for pyruvate dehydrogenase (E1) on the icosahedral acetyltransferase (E2) and the functional significance of this localization. The role(s) of PDPr on the catalytic activity, substrate specificity, and divalent cation requirements of PDP will be assessed using recombinant PDPc and PDPr and native PDH phosphatase: the function, if any of FAD in PDPr will be determined and the domain structure of PDPr will be delineated. Enzymic assays, equilibrium dialysis, limited proteolysis, and deletion mutagenesis will be used. Protein-protein interaction between E2, E3BP, and E1 will be assessed by protein engineering and binding studies in conjunction with cryoelectron microscopy and image reconstruction.